Synergistic Anticancer Effect Of The Total Saponine Of Aralia Elata Seem Leaves Combined With 5-Fluorouracil In Vitro

Objective:this experiment in human breast cancer MCF-7 cell as the research object,explores the liaodong Cong konoha total saponins in combination with 5-fluorouracil outside on mammary gland MCF-7 cell in the body whether have synergy effect,and further explore the possible mechanism of action,for the combination of the two medicine provides the theory basis for clinical treatment of breast cancer.Method:1.The determined by MTT method to study different concentrations in vitro proliferation liaodong Cong wood total saponins,5-fluorouracil with and MCF-7 to act on human breast cancer cells in vitro proliferation inhibition;2.Using the optical microscope cell in liaodong Cong konoha total soap and 5-fluorouracil to act on human breast cancer cells after MCF-7 cell morphological changes caused by;3.The use of Hoechst 33258 fluorescent staining to observe.cells in liaodong Cong konoha total saponins and 5-fluorouracil to act on human breast cancer cells after MCF-7 morphological changes caused by the nucleus;4.Flow cytometry analysis liaodong Cong konoha total saponins and 5-fluorouracil to act on human breast cancer cells after the MCF-7 caused by changes in the rate of apoptosis;5.ELISA kits liaodong Cong konoha total saponins and 5-fluorouracil to act on human breast cancer cells MCF-7 after training in the supernatant cytokine VEGF,TAF,and the change of MMP-2;6.Western blot method to detect the liaodong Cong konoha total saponins and 5-fluorouracil to act on human breast cancer cells apoptosis protein after MCF-7 caspase 3,and the change of caspase-7;7.Nude mouse transplantation tumor model,observe the liaodong Cong konoha total saponins and 5-fluorouracil used alone and in combination on breast cancer cell growth inhibition;8.HE dyeing observation of liaodong Cong konoha total saponins and 5-fluorouracil after separate and joint action in nude mice transplantation tumor pathological changes the results;9.ELISA kits liaodong Cong konoha total saponins and 5-fluorouracil separate and joint action in nude mice after cytokines in serum IL-2 and TNF alpha,IFN-gamma,TGF-beta,and the change of VEGF.The result:1.Determined by MTT experiment in vitro proliferation test results shows that:the liaodong Cong konoha total saponins and MCF-7 5-fluorouracil use in breast cancer cells growth inhibition,cell inhibitory effect with increase of drug concentration increased,the concentration dependence,and combination with synergy;2.Optical microscope MCF-7 cell:visible MCF-7 cell growth vigorous blank control group,and sharp.Liaodong Cong konoha total saponins group cell shrinkage,refraction sex is abate,5-fluorouracil group cells become circular,stick a wall is not tight,with floating cells and intercellular space greatens;Combination group is a large number of cells and cell debris floating,cytoplasm in the small particles of black dot,showed signs of cell apoptosis significantly;3.by fluorescence microscope after drug treatment of MCF-7 cell nucleus morphology results suggest:no drug control group,the nuclear shape round,uniform dispersion of light blue fluorescence.Liao dong Cong konoha total saponins and separate treatment group and combined treatment group of 5 fluorouracil MCF-7 cell nucleus or cytoplasm with strong fluorescence dye dense granules patches,the nucleus pycnosis,fracture,show the chromatin in the nucleus morphological changes have taken place,showing a typical apoptosis characteristics;4.Flow cytometry results showed that the liaodong Cong konoha total saponins,5-fluorouracil joint application group compared with the medicine group cell apoptosis rate increased significantly,and the apoptosis in advance to early apoptosis,the difference was statistically significant,prompt combination effect on promoting apoptosis than medication alone;5.ELISA test,according to the results of liaodong Cong konoha total saponins with 5-fluorouracil separate and joint function after cell culture supernatant of VEGF,TAF and MMP-2 content decreased,the combined treatment group decreased significantly(p<0.05);Caspase 36.Western blot method and the result of caspase-7 protein expression tip:compared with the blank control group,liaodong Cong konoha total saponins with 5-fluorouracil separate protein expression and combination group were cut,combination group decreased significantly;7.In vivo experiments showed liaodong Cong konoha total saponins group,5-fluorouracil and two drugs combination group on MCF-7 nude mouse transplantation tumor growth inhibition rate was 29.01%,16.79%and 63.36%respectively,liaodong Cong konoha total saponins and 5-fluorouracil is compared with the single joint group was obviously inhibit the growth of tumor cells(p<0.05);8.Tumor tissue pathological slices of HE staining,light microscope observation results showed that under the blank group closely packed cells,tumor cells is given priority to with round or oval,larger nucleus.Liaodong Cong konoha total saponins,5 fluorouracil and the joint group of cancer cell size is not uniform,the two drugs were under water degeneration,the nucleus pycnosis,dissolve and cancer cell disruption,tumor tissue degeneration,liaodong Cong konoha total saponins group necrosis is serious;Combination group of apoptosis significantly.9.ELISA test results show that:the liaodong Cong konoha total saponins and 5-fluorouracil separate and combined the two batches of nude mouse transplantation tumor model in serum,compared with the blank control group and combination group alone TNF alpha increased and the content of IFN-gamma,IL-2,TGF-beta,and the content of VEGF decreased,and the combination group was significantly.Conclusion:1.liaodong Cong konoha total saponins and 5-fluorouracil combination on human breast cancer cell growth inhibition of MCF-7 have synergy,and presents the concentration dependence.2.liaodong Cong konoha total saponins and used in combination with 5-fluorouracil can increase the rate of apoptosis,and apoptosis in advance to the early,the apoptosis mechanism may be through lower VEGF,TAF and MMP-2 expression.Two medicine joint is bigger than used alone can influence the expression of apoptosis related proteins,activating caspase-and caspase 3-7,have stronger degree of apoptosis.3.liaodong Cong konoha total saponins and used in combination with 5-fluorouracil in human breast cancer in vivo transplantation tumor animal models on has made a show of synergy,to slow tumor growth and promote tumor necrosis,at the same time within the influence of serum IL-2 and TNF alpha,IFN-gamma,TGF-beta and VEGF expression of cytokines.